Texturizing lactic acid bacteria strains

ABSTRACT

The present invention relates to mutants of lactic acid bacteria which are resistant towards the antibiotic D-cycloserine and/or functionally equivalent antibiotics and which were found to give an increased texture when grown in milk while maintaining the other growth properties of the parent strain. The present invention, furthermore, relates to compositions comprising such mutants, and to dairy products fermented with the lactic acid bacteria resistant towards D-cycloserine and/or functionally equivalent antibiotics.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/880,620, filed Apr. 19, 2013, which is the U.S. NationalStage of International Application No. PCT/EP2011/068478 filed Oct. 21,2011, which was published on Apr. 26, 2012, as WO 2012/052557, whichclaims the benefit of EP Application No. 10188511.9, filed Oct. 22,2010. The respective contents of these applications are incorporatedhere by reference in their entirety.

FIELD OF INVENTION

The present invention relates to mutants of lactic acid bacteria, suchas Lactobacillus delbrueckii subsp bulgaricus and Streptococcusthermophilus, which are resistant towards the antibiotic D-cycloserineand/or functionally equivalent antibiotics, and which were found to givean increased texture when grown in milk while maintaining the othergrowth properties of the parent strain. The present invention,furthermore, relates to cultures, such as starter cultures, comprisingsuch mutants, and to dairy products fermented with the cultures.

BACKGROUND OF INVENTION

The food industry uses numerous bacteria, in particular lactic acidbacteria, in order to improve the taste and the texture of foods butalso in order to extend the shelf life of these foods. In the case ofthe dairy industry, lactic acid bacteria are used intensively in orderto bring about the acidification of milk (by fermentation) but also inorder to texturize the product into which they are incorporated.

Among the lactic acid bacteria used in the food industry, there can bementioned the genera Streptococcus, Lactococcus, Lactobacillus,Leuconostoc, Pediococcus and Bifidobacterium. The lactic acid bacteriaof the species Streptococcus thermophilus are used extensively alone orin combination with other bacteria such as Lactobacillus delbrueckiisubsp bulgaricus for the production of food products, in particularfermented products. They are used in particular in the formulation ofthe ferments used for the production of fermented milks, for exampleyoghurts. Certain of them play a dominant role in the development of thetexture of the fermented product. This characteristic is closely linkedto the production of polysaccharides.

The current trend in yoghurts is for mild flavor and high texture. Todaythis is achieved by the use of cultures which produce a mild flavor andthe addition of thickeners or protein to give the desired thickness.Yoghurt producers would like to be able to make yoghurt with theseproperties without the addition of thickening agents. This will helpthem reduce cost and give a cleaner label. One very attractive way toachieve this would be to have a starter culture which produces a highlevel of texture.

In order to meet the requirements of the industry, it has becomenecessary to provide novel texturizing strains of lactic acid bacteria,in particular of Lactobacillus delbrueckii subsp bulgaricus andStreptococcus thermophilus, for texturizing food products. Especiallythere is a need for novel texturizing strains of Lactobacillusdelbrueckii subsp bulgaricus which can be used together with noveltexturizing strains of Streptococcus thermophilus.

Mutants resistant towards D-cycloserine have been described for a numberof different bacteria but it has never been reported that these mutantscan be used for increasing the texture of a dairy product. NeitherStreptococcus thermophilus nor Lactobacillus delbrueckii subspbulgaricus mutants have been described.

SUMMARY OF THE INVENTION

The inventors of the present invention have surprisingly found that agroup of lactic acid bacteria mutants resistant towards D-cycloserinegenerates higher shear stress and/or gel stiffness than the motherstrain when the bacteria are used for fermenting milk.

The present inventors have provided a method for obtaining suchtexturizing lactic acid bacteria strains which are resistant towards theantibiotic D-cycloserine and/or functionally equivalent antibiotics andthey have provided a novel group of D-cycloserine resistant lactic acidbacteria strains of the species Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus with high texturizingproperties.

In accordance with the above surprising findings, the present inventionrelates to texturizing lactic acid bacteria strains, such asLactobacillus delbrueckii subsp bulgaricus and Streptococcusthermophilus strains, which are D-cycloserine resistant and/or resistantto a functionally equivalent antibiotic and a method for obtaining suchstrains. Furthermore, the present invention relates to cultures, such asstarter cultures, comprising the mutants and to dairy products, such asfermented milk products fermented with the cultures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows acidification profiles (pH as a function of time) in milkat 43° C. for mother strain Lactobacillus delbrueckii subsp. bulgaricusCHCC13995 and two D-cycloserine mutants of CHCC13995 named CHCC12944 andCHCC12945.

FIG. 2 depicts shear stress (in 300 l/s (Pa)) in full fat milk with 2%skim milk powder at 43° C. for mother strain Lactobacillus delbrueckiisubsp. bulgaricus CHCC13995 and two D-cycloserine mutants of CHCC13995named CHCC12944 and CHCC12945.

FIG. 3 depicts gel stiffness (in 300 l/s (Pa)) in full fat milk with 2%skim milk powder at 43° C. for mother strain Lactobacillus delbrueckiisubsp. bulgaricus CHCC13995 and two D-cycloserine mutants of CHCC13995named CHCC12944 and CHCC12945.

FIG. 4 depicts shear stress (in 300 l/s (Pa)) in full fat milk with 2%skim milk powder at 43° C. for mother strain Streptococcus thermophilusCHCC13994 and 2 D-cycloserine mutants of CHCC13994 named CHCC13235 andCHCC13236.

FIG. 5 depicts gel stiffness (in 300 l/s (Pa)) in full fat milk with 2%skim milk powder at 43° C. for mother strain Streptococcus thermophilusCHCC13994 and 2 D-cycloserine mutants of CHCC13994 named CHCC13235 andCHCC13236.

DETAILED DISCLOSURE Definitions

As used herein, the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid, acetic acid and propionic acid. Theindustrially most useful lactic acid bacteria are found within the order“Lactobacillales” which includes Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacteriumspp., Enterococcus spp. and Propionibacterium spp. Additionally, lacticacid producing bacteria belonging to the group of the strict anaerobicbacteria, bifidobacteria, i.e. Bifidobacterium spp., are generallyincluded in the group of lactic acid bacteria. These are frequently usedas food cultures alone or in combination with other lactic acidbacteria. Lactic acid bacteria, including bacteria of the speciesLactobacillus sp. and Streptococcus thermophilus, are normally suppliedto the dairy industry either as frozen or freeze-dried cultures for bulkstarter propagation or as so-called “Direct Vat Set” (DVS) cultures,intended for direct inoculation into a fermentation vessel or vat forthe production of a dairy product, such as a fermented milk product.Such cultures are in general referred to as “starter cultures” or“starters”.

The term “milk” is to be understood as the lacteal secretion obtained bymilking any mammal, such as cows, sheep, goats, buffaloes or camels. Ina preferred embodiment, the milk is cow's milk. The term milk alsoincludes protein/fat solutions made of plant materials, e.g. soy milk.

The term “milk substrate” may be any raw and/or processed milk maternalthat can be subjected to fermentation according to the method of theinvention. Thus, useful milk substrates include, but are not limited to,solutions/suspensions of any milk or milk like products comprisingprotein, such as whole or low fat milk, skim milk, buttermilk,reconstituted milk powder, condensed milk, dried milk, whey, wheypermeate, lactose, mother liquid from crystallization of lactose, wheyprotein concentrate, or cream. Obviously, the milk substrate mayoriginate from any mammal, e.g. being substantially pure mammalian milk,or reconstituted milk powder.

Preferably, at least part of the protein in the milk substrate isproteins naturally occurring in milk, such as casein or whey protein.However, part of the protein may be proteins which are not naturallyoccurring in milk.

Prior to fermentation, the milk substrate may be homogenized andpasteurized according to methods known in the art.

“Homogenizing” as used herein means intensive mixing to obtain a solublesuspension or emulsion. If homogenization is performed prior tofermentation, it may be performed so as to break up the milk fat intosmaller sizes so that it no longer separates from the milk. This may beaccomplished by forcing the milk at high pressure through smallorifices.

“Pasteurizing” as used herein means treatment of the milk substrate toreduce or eliminate the presence of live organisms, such asmicroorganisms. Preferably, pasteurization is attained by maintaining aspecified temperature for a specified period of time. The specifiedtemperature is usually attained by heating. The temperature and durationmay be selected in order to kill or inactivate certain bacteria, such asharmful bacteria. A rapid cooling step may follow.

“Fermentation” in the methods of the present invention means theconversion of carbohydrates into alcohols or acids through the action ofa microorganism. Preferably, fermentation in the methods of theinvention comprises conversion of lactose to lactic acid.

Fermentation processes to be used in production of fermented milkproducts are well known and the person of skill in the art will know howto select suitable process conditions, such as temperature, oxygen,amount and characteristics of microorganism(s) and process time.Obviously, fermentation conditions are selected so as to support theachievement of the present invention, i.e. to obtain a dairy product insolid or liquid form (fermented milk product).

In the present context, a yoghurt starter culture is a bacterial culturewhich comprises at least one Lactobacillus delbrueckii subsp bulgaricusstrain and at least one Streptococcus thermophilus strain. In accordanceherewith, a “yoghurt” refers to a fermented milk product obtainable byinoculating and fermenting milk with a composition comprising aLactobacillus delbrueckii subsp bulgaricus strain and a Streptococcusthermophilus strain.

In the present context, the term “shear stress” determines viscosity.Viscosity (unit is Pa s) is defined as Shear Stress (Pa)/Shear rate(1/s).

Shear stress value is reported as a standard herein at shear rate=300l/s. Sensory experiments have shown (data not shown) that the bestcorrelation between rheological measurements and sensory viscosity/mouththickness are found when using the viscosity measured at shear rate 300l/s.

In the present context, the term “functionally equivalent antibiotic”should be understood as an antibiotic with the same mode of action orthe same target as D-cycloserine, such as e.g. other inhibitors ofD-alanyl-D-alanine ligases, such as e.g. vancomycin (Tytgat et al.(2009), Curr. Med. Chem. 16(20): 2566.2580) and other inhibitors ofD-alanine racemase, such as e.g. O-carbamoyl-D-serine, alaphosphin andthe haloalanines (Holtje (2004), The Desk Encyclopedia of Microbiology:239-258).

The term “resistant towards D-cycloserine and/or a functionallyequivalent antibiotic” herein means that a particular mutated bacterialstrain is not killed, or killed significantly more slowly compared tothe corresponding non-mutated strain from which the mutated strain isderived in the presence of said antibiotic in the culture medium.Dependent on the concentration of the antibiotic compound in the culturemedium, resistance can also be reflected by altered growth properties ofthe mutated and non-mutated strains. For example, a low concentration ofthe antibiotic in the culture medium will prevent or significantlydecrease the growth of non-mutated strains while the growth of themutated strains is not affected. Non-mutated strains which can be usedas sensitive reference strains in the assessment of resistancepreferably include the strains CHCC13995 and CHCC13994.

Examples 1 and 2 of the present application exemplify the isolation ofmutant strains of Lactobacillus delbrueckii subsp. bulgaricus andStreptococcus thermophilus, respectively, which are resistant toD-cycloserine. Based on the methods referred to in Example 1 and 2, theskilled person will be readily able to choose for each non-mutatedmother strain an amount of D-cycloserine or an amount of a functionallyequivalent antibiotic which is effective in killing the majority of thecells of the mother strain while not killing or killing significantlymore slowly mutant strains resistant towards D-cycloserine and/or afunctionally equivalent antibiotic.

In the present context, the term “mutant” should be understood as astrain derived, or a strain which can be derived from a strain of theinvention (or the mother strain) by means of e.g. genetic engineering,radiation and/or chemical treatment. The mutant can also be aspontaneously occurring mutant. It is preferred that the mutant is afunctionally equivalent mutant, e.g. a mutant that has substantially thesame, or Improved, properties (e.g. regarding viscosity, gel stiffness,mouth coating, flavor, post acidification, acidification speed, and/orphage robustness) as the mother strain. Such a mutant is a part of thepresent invention. Especially, the term “mutant” refers to a strainobtained by subjecting a strain of the invention to any conventionallyused mutagenization treatment including treatment with a chemicalmutagen such as ethane methane sulphonate (EMS) orN-methyl-N′-nitro-N-nitroguanidine (NTG), UV light, or to aspontaneously occurring mutant. A mutant may have been subjected toseveral mutagenization treatments (a single treatment should beunderstood one mutagenization step followed by a screening/selectionstep), but it is presently preferred that no more than 20, or no morethan 10, or no more than 5, treatments (or screening/selection steps)are carried out. In a presently preferred mutant less than 1%, less than0.1, less than 0.01, less than 0.001% or even less than 0.0001% of thenucleotides in the bacterial genome have been replaced with anothernucleotide, or deleted, compared to the mother strain.

In the present context, the term “variant” should be understood as astrain which is functionally equivalent to a strain of the invention,e.g. having substantially the same, or improved, properties e.g.regarding viscosity, gel stiffness, mouth coating, flavor, postacidification, acidification speed, and/or phage robustness). Suchvariants, which may be identified using appropriate screeningtechniques, are a part of the present invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising”, “having”, “including” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Implementation and Aspects of the Invention

D-cycloserine (D-4-amino-isoxazolidone) is an antibiotic which inhibitsalanine racemase, D-alanyl-D-alanine ligase, D-alanylalanine synthaseand D-alanine permease causing cell lysis. D-alanine racemase isessential for the production of D-alanine, an integral part of thepeptidoglycan layer of the cell wall. Mutants of Mycobacterium which areresistant to D-Cycloserine overproduce D-alanine by increasing theexpression of the D-alanine racemase gene (Caceres et al. (1997), J.Bacteriol. 179: 5046-5055). Thus, the inventors of the present inventioncontemplated that it would be possible to isolate spontaneous mutants oflactic acid bacteria strains which naturally have a higher production ofD-alanine. Such mutants were isolated and characterized.

A person of skill in the art will recognize that other antibiotics withthe same mode of action or the same target as D-cycloserine can be usedalone or in combination with D-cycloserine to isolate mutants of thetype described here. The present invention, therefore, also encompassesthe use of such other functionally equivalent antibiotics, such as otherinhibitors of D-alanyl-D-alanine ligases or O-alanine racemase.

The term “functionally equivalent antibiotics to D-cycloserine” as usedherein includes any compound which interferes with the synthesis ofD-alanine, with the transport of D-alanine into the cell, with theformation of D-alanyl-Dalanine from D-alanine or with the inclusion ofD-alanyl-D-alanine into building blocks of the peptidoglycan polymer.Such antibiotics include, but are not limited to, antibiotics thatinhibit the enzymes D-alanine-racemase, D-alanyl-D-alanine ligase,D-alanylalaninse synthase and D-alanine permease. Such functionallyequivalent antibiotics to D-cycloserine include, but are not limited to,vancomycin, O-carbamoyl-D-serine, alaphosphin and the haloalanines.

Some mutants of Lactobacillus delbrueckii subsp bulgaricus andStreptococcus thermophilus which are resistant to D-cycloserine werefound to give an increased texture when grown in milk compared to theparent strain while maintaining the other growth properties of theparent strain.

In accordance with the above surprising findings, the present inventionin a first aspect relates to a method for obtaining a texturizing lacticacid bacteria strain said method comprising:

-   -   a) providing a lactic acid bacteria mother strain;    -   b) isolating a mutant of the mother strain which is resistant        towards D-cycloserine and/or a functionally equivalent        antibiotic to D-cycloserine; and    -   c) selecting the mutant if it produces more texture, as        determined by greater shear stress and/or greater gel stiffness,        when grown in milk than the mother strain, thereby obtaining a        texturising lactic acid bacteria strain.

Thus, the first aspect relates to a method for obtaining a lactic acidbacteria mutant which results in increased shear stress and or/gelstiffness when grown in milk, said method comprising:

-   -   a) providing a lactic acid bacteria mother strain;    -   b) isolating a mutant of the mother strain which is resistant        towards D-cycloserine and/or a functionally equivalent        antibiotic to D-cycloserine; and    -   c) selecting the mutant if it produces more texture, as        determined by greater shear stress and/or greater gel stiffness,        when grown in milk than the mother strain, thereby obtaining a        lactic acid bacteria mutant which results in increased shear        stress and/or gel stiffness when grown in milk.

The production by a selected mutant of more texture than the motherstrains, as determined by greater shear stress and/or greater gelstiffness, occurs both in full fat and low-fat milk.

In a preferred embodiment the milk is cow milk. Preferably, the milk isfull-fat cow milk.

In a preferred embodiment the lactic acid bacteria mother strain is alactic acid bacteria selected from the group consisting of Lactobacillusdelbrueckii subsp bulgaricus and Streptococcus thermophilus. However,use of other lactic acid bacteria, such as bacteria of Lactococcus spp.,Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcusspp., Brevibacterium spp., Enterococcus spp., Propionibacterium spp.,and Bifidobacterium spp., are also part of the present invention.

In one preferred embodiment, mutants are obtained by incubating thelactic acid bacteria mother strain in/on growth medium containingD-cycloserine and/or a functionally equivalent antibiotic toD-cycloserine and isolating a mutant of the mother strain which iseither not killed by the D-cycloserine and/or the functionallyequivalent antibiotic to D-cycloserine or which is killed significantlymore slowly than the majority of the non-mutated cells tested. However,the present invention encompasses other ways known to the skilled personof isolating mutants resistant to D-Cycloserine and/or functionallyequivalent antibiotics to D-cycloserine. Accordingly, the mother strainused in the method of the first aspect of the present invention is astrain that is not resistant (or less resistant) to D-Cycloserine and/orthe functionally equivalent antibiotic to D-cycloserine.

In one embodiment the mutant may be a spontaneous mutant resistant toD-cycloserine and/or a functionally equivalent antibiotic toD-cycloserine.

In another embodiment the method of the invention may comprise a step ofmutagenising (e.g. by chemical treatment or radiation treatment, or bymeans of genetic engineering techniques) the mother strain, e.g. beforeor during step b.

Thus, in one embodiment the mutant may be obtained by mutagenesis of themother strain by means of e.g. chemical treatment or radiationtreatment, or a mutant obtained by means of genetic engineeringtechniques.

By “texture” or “mouthfeel” are meant the product's physical andchemical interaction in the mouth.

Methods for determining the texture of milk include measuring the shearstress (viscosity) or gel stiffness of the fermented milk and arereadily available and known in the art and exemplified herein.

The mutant is selected, in step c), if it produces more texture, asdetermined by greater shear stress and/or greater gel stiffness, infermented milk than the mother strain when inoculated in the same amountas the mother strain, such as in an amount of at least 10⁴ cells per mi.

In a preferred embodiment, the mutant generates a viscosity in milkmeasured as shear stress in 300 l/s (Pa) which is at least about 5%higher, at least about 10% higher, at least about 15% higher, at leastabout 20% higher, at least about 25% higher, at least about 30% higher,at least about 35% higher, at least about 40% higher, at least about 50%higher, than the viscosity generated by the mother strain.

In another preferred embodiment, the mutant generates a gel stiffness inmilk which is at least about 5% higher, at least about 10% higher, atleast about 15% higher, at least about 20% higher, at least about 25%higher, at least about 30% higher, at least about 35% higher, at leastabout 40% higher, at least about 50% higher, than the gel stiffnessgenerated by the mother strain.

Preferably, the mutant generates a viscosity in full fat cow milk added2% skim milk powder greater than about 105 Pa measured as shear stressin 300 l/s (Pa) after 12 hours of growth in the milk at 37° C. wheninoculated in an amount of at least 10⁴ cells per ml of milk in the caseof Lactobacillus delbrueckii subsp. bulgaricus strains and greater thanabout 75 Pa measured as shear stress in 300 l/s (Pa) after 12 hours ofgrowth in the milk at 37° C. when inoculated in an amount of at least10⁴ cells per ml of milk in the case of Streptococcus thermophilusstrains.

In a preferred embodiment, the mutant generates a gel stiffness in fullfat cow milk added 2% skim milk powder greater than about 80 Pa after 12hours of growth in the milk at 37° C. when inoculated in an amount of atleast 10⁴ cells per ml of milk in the case of Lactobacillus delbrueckiisubsp. bulgaricus strains and greater than about 140 Pa after 12 hoursof growth in the milk at 37° C. when inoculated in an amount of at least10⁴ cells per ml of milk in the case of Streptococcus thermophilusstrains.

In a second aspect the present invention is directed to a lactic acidbacteria strain obtainable by the method according to the first aspectof the invention.

In a preferred embodiment the lactic acid bacteria strain is a strainselected from the group consisting of Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus. In another preferredembodiment the lactic acid bacteria strain generates a viscosity in fullfat cow milk added 2% skim milk powder greater than about 105 Pameasured as shear stress in 300 l/s (Pa) after 12 hours of growth in themilk at 37° C. when inoculated in an amount of 10′ cells per ml of milkin the case of Lactobacillus delbrueckii subsp. bulgaricus strains andgreater than about 75 Pa measured as shear stress in 300 l/s (Pa) after12 hours of growth in the milk at 37° C. when inoculated in an amount ofat least 10⁴ cells per ml of milk in the case of Streptococcusthermophilus strains.

Preferably, the lactic acid bacteria strain generates a gel stiffness infull fat cow milk added 2% skim milk powder greater than about 80 Paafter 12 hours of growth in the milk at 37° C. when inoculated in anamount of at least 10⁴ cells per ml of milk in the case of Lactobacillusdelbrueckii subsp. bulgaricus strains and greater than about 140 Paafter 12 hours of growth in the milk at 37° C. when inoculated in anamount of at least 10⁴ cells per ml of milk in the case of Streptococcusthermophilus strains.

In a third aspect the present invention relates to a lactic acidbacteria strain belonging to the species Lactobacillus delbrueckiisubsp. bulgaricus, selected from the group consisting of: strainCHCC12944, that was deposited with Deutsche Sammlung von Mikroorganimenund Zellkulturen under accession number DSM 24019, strain CHCC12945,that was deposited with Deutsche Sammlung von Mikroorganismen undZellkulturen under accession number DSM 24020, and mutants and variantsof any of these. The mutants and variants show the shear stress and/orgel stiffness characteristics of the deposited strains from which theyhave been derived.

In a fourth aspect the present invention relates to a lactic acidbacteria strain belonging to the species Streptococcus thermophilus,selected from the group consisting of: strain CHCC13235, that wasdeposited with Deutsche Sammlung von Mikroorganismen und Zellkulturenunder accession number DSM 24010, strain CHCC13236, that was depositedwith Deutsche Sammlung von Mikroorganismen und Zellkulturen underaccession number DSM 24011, and mutants and variants of any of these.The mutants and variants show the shear stress and/or gel stiffnesscharacteristics of the deposited strains from which they have beenderived.

In a fifth aspect the present invention relates to a lactic acidbacteria strain belonging to the species Lactobacillus delbrueckiisubsp. bulgaricus resistant to D-cycloserine and/or a functionallyequivalent antibiotic having the same or improved gel stiffness and/orshear stress characteristics of strain CHCC12944, that was depositedwith the Deutsche Sammlung von Mikroorganismen und Zellkulturen underaccession number DSM 24019 or strain CHCC12945, that was deposited withthe Deutsche Sammlung von Mikroorganismen und Zellkulturen underaccession number DSM 24020.

In a sixth aspect the present invention concerns a lactic add bacteriastrain belonging to the species Streptococcus thermophilus resistant toD-cycloserine and/or a functionally equivalent antibiotic having thesame or improved gel stiffness and/or shear stress characteristics ofstrain CHCC13235, that was deposited with the Deutsche Sammlung vonMikroorganismen und Zellkulturen under accession number DSM 24010 orstrain CHCC13236, that was deposited with the Deutsche Sammlung vonMikroorganismen und Zellkulturen under accession number DSM 24011.

In a seventh aspect the present invention concerns a compositioncomprising a lactic acid bacteria strain according to the second tosixth aspects of the present invention.

In one embodiment of the present invention the composition comprises,either as a mixture or as a kit-of-parts:

-   -   i) a strain belonging to the species Streptococcus thermophilus        according to the second, fourth or sixth aspect of the        invention; and    -   ii) a strain belonging to the species Lactobacillus delbrueckii        subsp. bulgaricus according to the second, third or fifth aspect        of the invention.

The composition in one embodiment preferably comprises at least 10⁹ CFU(cell forming units) of a strain belonging to the species Lactobacillusdelbrueckii subsp bulgaricus; and at least 10¹⁰ CFU of a strainbelonging to the species Streptococcus thermophilus. The composition inanother embodiment comprises at least 10¹¹ CFU (cell forming units) of astrain belonging to the species Lactobacillus delbrueckii subspbulgaricus; and at least 10¹² CFU of a strain belonging to the speciesStreptococcus thermophilus.

The composition in one preferred embodiment may advantageously be usableas a starter culture, and is in frozen, freeze-dried or liquid form.

The present invention in an eighth aspect relates to a method ofproducing a dairy product, such as a fermented milk product, comprisingfermenting a milk substrate with a lactic acid bacteria strain accordingto any of the second to sixth aspects of the present invention or acomposition according to the seventh aspect of the present invention,thereby obtaining a dairy product.

The dairy product produced by this method thereby comprises the lacticacid bacteria strain according to any of the second to sixth aspects ofthe present invention or the lactic acid bacteria strains of thecomposition according to the seventh aspect of the present invention,respectively.

In the method, the milk substrate may be fermented with a strain of theinvention, such as a strain belonging to the species Streptococcusthermophilus before, during, or after the fermentation with a strain ofthe invention, such as a strain belonging to a Lactobacillus species.

Further, the method may include adding an enzyme to the milk substratebefore, during and/or after the fermenting, such as an enzyme selectedfrom the group consisting of: an enzyme able to crosslink proteins,transglutaminase, an aspartic protease, chymosin, and rennet.

In a ninth aspect the present invention concerns a dairy product, suchas a fermented milk product (e.g. yoghurt or buttermilk) or a cheese(e.g. fresh cheese of pasta filata), obtainable by a method according tothe eight aspect of the invention.

The dairy product comprises lactic acid bacteria.

The dairy product may optionally comprise an ingredient selected fromthe group consisting of: a fruit concentrate, a syrup, a probioticbacterial culture, a coloring agent, a thickening agent, a flavoringagent, and a preserving agent; and/or which optionally is in the form ofa starred type product, a set type product, or a drinkable product.

In a tenth aspect the present invention relates to a dairy productcomprising a lactic acid bacteria strain according to any of the secondto sixth aspects of the present invention.

In an eleventh aspect the present invention relates to use of a lacticacid bacteria strain according to any of the second to sixth aspects formanufacture of a fermented milk product.

The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Use of D-Cycloserine to Isolate Mutants inLactobacillus delbrueckii Subsp. Bulgaricus with Improved RheologicalProperties

Materials and Methods

Strains

Lactobacillus delbrueckii subsp. bulgaricus CHCC13995

Lactobacillus delbrueckii subsp. bulgaricus CHCC12944 (D-cycloserineresistant mutant of CHCC13995)

Lactobacillus delbrueckii subsp. bulgaricus CHCC12945 (D-cycloserineresistant mutant of CHCC13995)

Mutant Isolation

In order to isolate mutants of the Lactobacillus delbrueckii subsp.bulgaricus strain CHCC13995, cells derived from the growth of a singlecolony were inoculated into MRS broth containing in the range of 50-100μg/ml D-cycloserine and grown to saturation. Cells were diluted andplated on MRS plates and colonies were screened in microtiter plates forthe ability to grow in the presence of in the range of 50-100 μg/mlD-cycloserine. Typically, 25% of the resulting colonies were identifiedas fast growers in the presence of D-cycloserine. These were chosen forfurther study. The selected D-cycloserine resistant mutants were furtherpurified and tested for their ability to grow in milk. During this workit was observed that some of the mutants produced considerably moretexture than the parent strain under these conditions.

Rheology

A rheological analysis was done on a StressTech rheometer from ReologicaInstruments AB, Sweden, following growth in full fat cow milk with 2%added skim milk powder.

Results

Two mutant derivatives of CHCC13995, designated CHCC12944 and CHCC12945,produced significantly more texture with an Increased shear stress(viscosity) as well as gel stiffness than the mother strain. This wasvery evident just by stirring a pipette in the fermented milk.Furthermore, it was observed that the amount of whey differed betweenthe various mutants and this became one of the parameters used to selectthe mutants for further study (see Table 1 below). The mutants selectedwere those which showed a low level of syneresis: mutant 1 and mutant 11in the example below (Table 1) corresponds to mutants CHCC12944 andCHCC12945, respectively.

TABLE 1 Mutant 1 and mutant 11 were chosen among the 20 mutants, becauseof the low volume of whey produced. The empty lanes are mutants that didnot coagulate milk. The 2 mutants were designated CHCC12945 andCHCC12944, respectively. Mut. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 ml 4 16 — 17 8 8 10.5 22 — 16 7 5 15 32 11 — 16 20 — — ml wheyafter growth for 24 hours in 100 ml B-milk at 43° C.

The actual rheology tests support the observation of increased thickness(Table 2 and FIGS. 2 and 3). The increase in gel stiffness of themutants CHCC12945 and CHCC12944 is between 52% and 59%. Shear stress isalso higher but not as much as for the gel stiffness (Table 2 and FIGS.2 and 3).

TABLE 2 Sample Shear stress at 300 1/s (Pa) Gel stiffness at 1 HZ (Pa)CHCC13995 99 58.55 CHCC12945 108.5 93.20 CHCC12944 108 89.05

One thing is improved rheology but the mutants would be less attractiveif other important properties such as acidification were affected. Theacidification profile when growing in full fat cow milk with additional2% skim milk powder at 43° C. is the same for the chosen mutants as forthe relevant mother strain (FIG. 1).

Conclusion

The Lactobacillus delbrueckii subsp. bulgaricus D-cycloserine mutantsdescribed herein may be incorporated into a culture, such as a starterculture, which produces a desirable high level of texture.

Example 2 Use of D-Cycloserine to Isolate Mutants in Streptococcusthermophilus with Improved Rheological Properties

Materials and Methods

Strains

Streptococcus thermophilus CHCC13994

Streptococcus thermophilus CHCC13236 (D-cycloserine resistant mutant ofCHCC13994)

Streptococcus thermophilus CHCC13235 (D-cycloserine resistant mutant ofCHCC13994)

Mutant Isolation:

In order to isolate mutants of Streptococcus thermophilus CHCC13994.cells derived from the growth of a single colony were inoculated intoM17+2% lactose broth containing in the range of 50-70 μg/mlD-cycloserine and grown to saturation. Cells were diluted and plated onM17+2% lactose plates and colonies were picked and screened in microtiter plates for the ability to grow in the presence of in the range of50-70 μg/ml D-cycloserine. Typically, 25% of the resulting colonies wereidentified as fast growers in the presence of D-cycloserine. These werechosen for further study. The selected D-cycloserine resistant mutantswere further purified and tested for their ability to grow in milk.During this work it was observed if some of the mutants producedconsiderably more texture than the parent strain under these conditions.The amount of whey was also measure as indication of high or low gelstiffness.

Rheology

A rheological analysis was done on a StressTech rheometer from ReoLogicaInstruments AB, Sweden, following growth in full fat cow milk with 2%added skim milk powder.

Results

Two mutant derivatives of CHCC13994, designated CHCC13235 and CHCC13236,produced more texture than the mother strain. This was very evident justby stirring a pipette in the fermented milk. Furthermore, it wasobserved that the amount of whey differed between the various mutantsand this became one of the parameters used to select the mutants forfurther study (see Table 3 below). The mutants selected, no. 9 and no.13 in table 3, were two showing a low level of syneresis. The 2 mutantswere designated CHCC13236 and CHCC13235, respectively.

TABLE 3 2 mutants no. 9 and no. 13 were chosen among the 20 mutants.Mut. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ml 12 20 10 5.57 10 7.5 12 7 22 6.5 — 6.5 7 7.5 6 10 10 5 6 ml whey after growth for 24hours in 50 ml B-milk at 37° C.

The actual rheology tests support the observation of increased thickness(FIG. 4 and FIG. 5). The mutant CHCC13236 had a 15% increase in shearstress and 19% in gel stiffness, where as mutant CHCC13235 only showedan increase in gel stiffness (23%) (Table 4).

TABLE 4 Sample Shear stress at 300 1/s (Pa) Gel stiffness at 1 HZ (Pa)CHCC13994 69.6 136.6 CHCC13236 79.7 162.5 CHCC13235 64.9 167.3

Regarding acidification, the profile is the same for the chosen mutantsas for the relevant another strain (data not shown).

Conclusion

The Streptococcus thermophilus D-cycloserine mutants described hereinmay be incorporated into a culture, such as a starter culture, whichproduces a desirable high level of texture.

Example 3 Use of S thermophilus and Lb. Delbrueckii Subsp BulgaricusD-Cycloserine Mutants for Preparation of a Fermented Milk Product

Yoghurt was made with a combination of S. thermophilus and Lb.delbrueckii subsp bulgaricus, mixed at a 9:1 ratio and inoculated intofull fat cow milk with 2% added skim milk powder. One mixture comprisedthe parent strains CHCC13994 and CHCC13995; a second comprised S.thermophilus D-cycloserine mutant CHCC13236 and Lb. delbrueckii subspbulgaricus D-cycloserine mutant CHCC12945 and a third comprised S.thermophilus D-cycloserine mutant CHCC13235 and Lb. delbrueckii subspbulgaricus D-cycloserine mutant CHCC12945. Comparing the acidificationprofiles between the three combinations showed that the mixtures withtwo mutants had a slightly higher final pH than the combination with theparent strains (data not shown). Stirring with a spoon showed that theproducts made with the D-cycloserine mutants had a noticeably increasedresistance to stirring compared to the product with the parent strains.Additionally, the resulting yoghurt was considered to have a more creamytexture and a fresher taste when evaluated by three people.

Deposits and Expert Solution

The strains of Lactobacillus delbrueckii subsp. bulgaricus CHCC12944,CHCC12945 and CHCC13995 were deposited at DSMZ (Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124Braunschweig, Germany) under the accession numbers DSM 24019, DSM 24020,and DSM 24021, respectively, on 22 Sep. 2010.

The strains of Streptococcus thermophilus CHCC13235, CHCC13236, andCHCC13994 were deposited at DSMZ (Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany)under the accession numbers DSM 24010, DSM 24011, and DSM 24012,respectively, on 22 Sep. 2010.

The deposits have been made under the conditions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure.

The Applicant requests that a sample of the deposited microorganismsshould be made available only to an expert approved by the Applicant.

REFERENCES

-   Tytgat et al. (2009), Curr. Med. Chem. 16(20): 2566-2580.-   Caceres et al. (1997), 3. Bacteriol. 179: 5046-5055.-   Holtje (2004), The Desk Encyclopedia of Microbiology: 239-258.

All references cited in this patent document are hereby incorporatedherein in their entirety by reference.

The invention claimed is:
 1. A texturizing lactic acid bacteria strainselected from the group consisting of: (a) the D-cycloserine resistantLactobacillus delbrueckii subsp bulgaricus strain CHCC12944 depositedwith the Deutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ)under accession number DSM 24019 and mutant strains derived therefromthat are resistant to D-cycloserine and produce the same or more textureas the deposited strain as determined by shear stress and/or gelstiffness when inoculated into fermented milk in the same amount of atleast 10⁴ cells/ml as the deposited strain; (b) the D-cycloserineresistant Lactobacillus delbrueckii subsp bulgaricus strain CHCC12945deposited with the DSMZ under accession number DSM 24020 and mutantstrains derived therefrom that are resistant to D-cycloserine andproduce the same or more texture as the deposited strain as determinedby shear stress and/or gel stiffness when inoculated into fermented milkin the same amount of at least 10⁴ cells/ml as the deposited strain; (c)the D-cycloserine resistant Streptococcus thermophilus strain CHCC13235that was deposited with the DSMZ under accession number DSM 24010 andmutant strains derived therefrom that are resistant to D-cycloserine andproduce the same or more texture as the deposited strain as determinedby shear stress and/or gel stiffness when inoculated into fermented milkin the same amount of at least 10⁴ cells/ml as the deposited strain; and(d) the D-cycloserine resistant Streptococcus thermophilus strainCHCC13236 deposited with the DSMZ under accession number DSM 24011 andmutant strains derived therefrom that are resistant to D-cycloserine andproduce the same or more texture as the deposited strain as determinedby shear stress and/or gel stiffness when inoculated into fermented milkin the same amount of at least 10⁴ cells/ml as the deposited strain. 2.The texturizing lactic acid bacteria strain of claim 1, selected fromthe group consisting of the D-cycloserine resistant Lactobacillusdelbrueckii subsp bulgaricus strain CHCC12944 deposited with theDeutsche Sammlung von Mikroorganismen and Zellkulturen (DSMZ) underaccession number DSM 24019 and mutant strains derived therefrom that areresistant to D-cycloserine and produce the same or more texture as thedeposited strain as determined by shear stress and/or gel stiffness wheninoculated into fermented milk in the same amount of at least 10⁴cells/ml as the deposited strain.
 3. The texturizing lactic acidbacteria strain of claim 1, selected from the group consisting of theD-cycloserine resistant Lactobacillus delbrueckii subsp bulgaricusstrain CHCC12945 deposited with the DSMZ under accession number DSM24020 and mutant strains derived therefrom that are resistant toD-cycloserine and produce the same or more texture as the depositedstrain as determined by shear stress and/or gel stiffness wheninoculated into fermented milk in the same amount of at least 10⁴cells/ml as the deposited strain.
 4. The texturizing lactic acidbacteria strain of claim 1, selected from the group consisting of theD-cycloserine resistant Streptococcus thermophilus strain CHCC13235 thatwas deposited with the DSMZ under accession number DSM 24010 and mutantstrains derived therefrom that are resistant to D-cycloserine andproduce the same or more texture as the deposited strain as determinedby shear stress and/or gel stiffness when inoculated into fermented milkin the same amount of at least 10⁴ cells/ml as the deposited strain. 5.The texturizing lactic acid bacteria strain of claim 1, selected fromthe group consisting of the D-cycloserine resistant Streptococcusthermophilus strain CHCC13236 deposited with the DSMZ under accessionnumber DSM 24011 and mutant strains derived therefrom that are resistantto D-cycloserine and produce the same or more texture as the depositedstrain as determined by shear stress and/or gel stiffness wheninoculated into fermented milk in the same amount of at least 10⁴cells/ml as the deposited strain.
 6. A composition comprising atexturizing lactic acid bacteria strain according to claim
 1. 7. Thecomposition according to claim 6, in frozen, freeze-dried or liquidform.
 8. The composition according to claim 6 comprising as a mixture oras separate components: (i) a Lactobacillus delbrueckii subsp bulgaricusstrain selected from the group consisting of (a) the D-cycloserineresistant strain CHCC12944 deposited with the Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ) under accession number DSM 24019and mutant strains derived therefrom that are resistant to D-cycloserineand produce the same or more texture as the deposited strain asdetermined by shear stress and/or gel stiffness when inoculated intofermented milk in the same amount of at least 10⁴ cells/ml as thedeposited strain and (b) the D-cycloserine resistant strain CHCC12945deposited with the DSMZ under accession number DSM 24020 and mutantstrains derived therefrom that are resistant to D-cycloserine andproduce the same or more texture as the deposited strain as determinedby shear stress and/or gel stiffness when inoculated into fermented milkin the same amount of at least 10⁴ cells/ml as the deposited strain; and(ii) a Streptococcus thermophilus strain selected from the groupconsisting of (c) the D-cycloserine resistant CHCC13235 that wasdeposited with the DSMZ under accession number DSM 24010 and mutantstrains derived therefrom that are resistant to D-cycloserine andproduce the same or more texture as the deposited strain as determinedby shear stress and/or gel stiffness when inoculated into fermented milkin the same amount of at least 10⁴ cells/ml as the deposited strain and(d) the D-cycloserine resistant strain CHCC13236 deposited with the DSMZunder accession number DSM 24011 and mutant strains derived therefromthat are resistant to D-cycloserine and produce the same or more textureas the deposited strain as determined by shear stress and/or gelstiffness when inoculated into fermented milk in the same amount of atleast 10⁴ cells/ml as the deposited strain.
 9. The composition accordingto claim 8, in frozen, freeze-dried or liquid form.
 10. A method ofproducing a dairy product, comprising fermenting a milk substrate with atexturizing lactic acid bacteria strain according to claim
 1. 11. Amethod of producing a dairy product, comprising fermenting a milksubstrate with a composition according to claim
 6. 12. A method ofproducing a dairy product, comprising fermenting a milk substrate with acomposition according to claim
 8. 13. A dairy product comprising atexturizing lactic acid bacteria strain according to claim 1.